The long-term objective of this research is to understand how the environmental bacterium Listeria monocytogenes (Lm) adapts to life within mammalian cells to become a human pathogen. Lm is a facultative intracellular bacterium that survives as a saprophyte in soil but is capable of transitioning into a pathogen upon entry into a mammalian host. As a pathogen, Lm remains an increasingly important agent of serious food- borne infections and has been responsible for some of the largest food safety recalls in U.S. history. Central to Lm's ability to cause disease is the regulated transport of virulence factors across the bacterial membrane and the rigid cell wall;this critical process has not been well characterized in Gram-positive bacteria. This proposal focuses on molecular and mechanistic analyses of PrsA2, a post-translocation secretion chaperone with peptidyl-prolyl cis/trans isomerase activity that is essential for Lm virulence. Lm strains lacking PrsA2 function are severely attenuated in mouse models of infection and exhibit reduced secretion and activity of at least two key Lm virulence gene products, the cholesterol-dependent cytolysin listeriolysin O (LLO) and the broad specificity phospholipase PC-PLC. Both of these gene products contribute to Lm's ability to gain access to the host cell cytosol and to spread of the bacteria into new host cells. Preliminary experiments indicate that PrsA2 directly regulates LLO stability, and that PrsA2 may be required for the secretion and/or activity of additional Lm factors with important roles in bacterial virulence. The experiments outlined within this proposal will define the role of PrsA2 in regulating the activity of Lm secreted proteins used to secure the bacterium's intracellular replication niche. Aim 1 will determine the molecular mechanism by which PrsA2 regulates the secretion and activity of LLO and PC-PLC. Aim 2 will examine a number of potential PrsA2 substrates with roles in bacterial virulence for direct PrsA2 interactions. Experiments will further determine the nature of a suppressor mutation that partially compensates for PrsA2 function. Aim 3 experiments focus on the molecular analysis of PrsA2 functional domains based on structural modeling of PrsA2. The ultimate goal of the specific aims will be to elucidate how PrsA2 modulates the secretion and/or activity of virulence factors to promote bacterial pathogenesis in mammalian hosts. PUBLIC HEALTH RELEVANCE: Listeria monocytogenes represents an increasingly significant health threat as it has been associated with several multi-state food-borne outbreaks that have resulted in thousands of illnesses and several hundred deaths within the past few years. The largest and most expensive (>$140 million) recalls of food products in history continue to occur as a result of L. monocytogenes contamination, and the bacterium generally ranks as the third or fourth most common cause of bacterial meningitis in North America. This proposal will elucidate how L. monocytogenes regulates the activity of secreted factors critical for bacterial virulence;this Information may result in the identification of new Gram-positive bacterial drug targets as well as enhance the development of L. monocytogenes-based vaccines for delivery of foreign antigens.